EP1323829B1 - Method for the preparation of protected, enantiomerically enriched cyanohydrines by in-situ derivatisation - Google Patents

Method for the preparation of protected, enantiomerically enriched cyanohydrines by in-situ derivatisation Download PDF

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EP1323829B1
EP1323829B1 EP02026849A EP02026849A EP1323829B1 EP 1323829 B1 EP1323829 B1 EP 1323829B1 EP 02026849 A EP02026849 A EP 02026849A EP 02026849 A EP02026849 A EP 02026849A EP 1323829 B1 EP1323829 B1 EP 1323829B1
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alkyl
formula
process according
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EP1323829A2 (en
EP1323829A3 (en
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Wolfgang Skranc
Peter Pöchlauer
Irma Wirth
Rudolf Neuhofer
Herbert Mayrhofer
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Patheon Austria GmbH and Co KG
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DSM Fine Chemicals Austria Nfg GmbH and Co KG
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/002—Nitriles (-CN)
    • C12P13/004—Cyanohydrins

Definitions

  • Cyanohydrins are for example for the synthesis of alpha-hydroxy acids, alpha-hydroxy ketones, beta-amino alcohols, which are used to obtain biologically active substances, eg. As pharmaceutical agents, vitamins or pyrethroid compounds find use of importance.
  • a cyanohydrin can be carried out by addition of hydrogen cyanide (HCN) to the carbonyl group of an aldehyde or a ketone, resulting in enantiomeric mixtures of asymmetric cyanohydrins.
  • HCN hydrogen cyanide
  • Many methods are based on carrying out the addition of HCN to the carbonyl group in the presence of a chiral catalyst, for example a hydroxynitrile lyase.
  • HCN is a very toxic substance, it is constantly trying to avoid direct use or direct handling.
  • Another cyanide group donor is, for example, trimethylsilyl cyanide, which according to J. Am. Chem. Soc. 2001, 123, 9908-9909 is reacted with sugar derivatives at -40 ° C in an absolute alcohol.
  • cyanohydrins are unstable per se and tend to decompose in reversal of their formation reaction, so they have already been tried by the various additives, in particular of acids such as sulfuric acid, phosphoric acid, HCl, toluenesulfonic acid, acetic acid, propionic acid, etc.
  • the object of the present invention was to find a hydroxynitrile lyyl catalyzed process for the preparation of stable, enantiomerically enriched cyanohydrins in which the direct use of hydrogen cyanide is avoided and which allows a shift in equilibrium to achieve high conversions.
  • this object could be achieved by a method in which carbonic acid nitriles are used as cyanide group donors, whereby an in situ derivatization and thus stabilization of the enantiomerically enriched cyanohydrins occurs.
  • the present invention accordingly provides a process for the preparation of protected, enantiomerically enriched cyanohydrins of the formula in the R 1 and R 2, independently of one another, an optionally mono- or polysubstituted C 1 -C 20 -alkyl, C 5 -C 20 -aryl, C 5 -C 20 -heteroaryl, C 5 -C 20 -alkaryl-C C 5 -C 20 -alkylheteroaryl or C 5 -C 20 -aralkyl radical or an optionally mono- or polysubstituted C 5 -C 20 -heterocycle or C 5 -C 20 -alkyl heterocycle or together an optionally substituted C 4 -C 20 -alkylene radical which may mean one or more heteroatoms in the chain, or one of the radicals Is hydrogen, and R3 may be an optionally substituted C 1 -C 20 alkyl, C 5 -C 20 aryl or C 5 -C 20 heteroary
  • R1 and R2 are each independently an unsubstituted, monosubstituted or polysubstituted C 1 -C 20 alkyl, C 5 -C 20 aryl, C 5 -C 20 -heteroaryl, C 5- C 20 -Alkaryl-, C 5 -C 20 - Alkylheteroaryl- or C 5 -C 20 -Aralkylrest or an optionally mono- or polysubstituted C 5 -C 20 heterocycle or C 5 -C 20 alkylheterocycle.
  • C 1 -C 20 -alkyl saturated or mono- or polyunsaturated, linear, branched or cyclic, primary, secondary or tertiary hydrocarbon radicals.
  • C 1 -C 20 -alkyl radicals such as, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, butenyl, butynyl, pentyl, cyclopentyl, isopentyl, neopentyl, pentenyl , Pentynyl, hexyl, isohexyl, cyclohexyl, cyclohexylmethyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, octyl, cyclooctyl, decyl, cyclodecyl, dodecyl, cycl
  • C 1 -C 12 -alkyl radicals and particularly preferably C 1 -C 8 -alkyl radicals.
  • the alkyl group may optionally be monosubstituted or polysubstituted by groups inert under the reaction conditions.
  • Suitable substituents are, for example, optionally substituted aryl or heteroaryl groups, such as phenyl, phenoxy or indolyl groups, halogen, hydroxy, hydroxy-C 1 -C 5 -alkyl, C 1 -C 6 -alkoxy, aryloxy, preferably C C 6 -C 20 -aryloxy, C 1 -C 6 -alkylthio, -amino, alkylamino, preferably C 1 -C 6 -alkylamino, arylamino, preferably C 6 -C 20 -arylamino, ether, Thioethers, carboxylic acid ester, carboxylic acid amide, sulfoxide, sulfonic, sulfonic, sulfonic acid, sulfinic, mercaptan, nitro or azido groups.
  • aryl or heteroaryl groups such as phenyl, phenoxy or indolyl groups,
  • Aryl is preferably C 6 -C 20 -aryl groups, such as phenyl, biphenyl, naphthyl, indenyl, fluorenyl, etc
  • the aryl group may optionally be monosubstituted or polysubstituted. Suitable substituents are in turn optionally substituted aryl or heteroaryl groups such as phenyl, phenoxy or indolyl, halogen, hydroxy, hydroxy-C 1 -C 5 alkyl, C 1 -C 6 alkoxy, aryloxy, preferred C 6 -C 20 -aryloxy, C 1 -C 6 -alkylthio, amino, alkylamino, preferably C 1 -C 6 -alkylamino, arylamino, preferably C 6 -C 20 -arylamino, ether, Thioethers, carboxylic acid ester, carboxylic acid amide, sulfoxide, sulfonic, sulfonic, sulfonic acid, sulfinic, mercaptan, nitro or azido groups.
  • aryl or heteroaryl groups such as phenyl, phenoxy
  • alkaryl or alkylaryl are meant alkyl groups having an aryl substituent.
  • Aralkyl or arylalkyl refers to an aryl group having an alkyl substituent.
  • heteroaryl or heterocycle are meant cyclic radicals containing at least one S, O or N atom in the ring. These are, for example, furyl, Pyridyl, pyrimidyl, thienyl, isothiazolyl, imidazolyl, tetrazolyl, pyrazinyl, benzofuranyl, benzothiophenyl, quinolyl, isoquinolyl, benzothienyl, isobenzofuryl, pyrazolyl, indolyl, isoindolyl, benzoimidazolyl, purinyl, carbazolyl, oxazolyl, thiazolyl, isothiazolyl, 1,2,4- Thiadiazolyl, isoxazolyl, pyrrolyl, quinazolinyl, pyridazinyl, phthalazinyl, morpholinyl, etc Functional O or N groups can be protected if necessary.
  • alkyl heteroaryl or alkyl heterocycle are meant alkyl groups which are substituted by a heteroaryl group or by a heterocycle.
  • R 1 and R 2 preferably denote a saturated, linear or branched C 1 -C 8 -alkyl radical, a benzyl radical or a phenyl radical, where the radicals are optionally mono- or polysubstituted by F, Cl, OH, carboxylic acid derivatives, such as carboxylic acid esters or carboxamides, amino, C 1 -C 6 alkylamino, C 6 -C 20 arylamino, C 1 -C 6 alkoxy, C 6 -C 20 aryloxy, or nitro may be substituted.
  • R 1 and R 2 together may also be an optionally substituted C 4 -C 20 -alkylene radical which contains one or more heteroatoms from the group consisting of O, N or S or an NR 4 R 5 -group, where R 4 and R 5 independently of one another are H or C 1 -C 6 - Alkyl may be included in the chain.
  • the starting materials are cyclic ketones.
  • the alkylene radical can still, in turn, depending on the ring size of the cyclic ketone still have one or two double bonds, which in a 5-membered ring may not be in conjugation to the carbonyl group.
  • the alkylene radical may also be substituted one or more times by the radicals mentioned above.
  • one of the radicals R 1 and R 2 may also be hydrogen.
  • the educts are aldehydes.
  • R3 is an optionally substituted C, C 5- C 20 aryl or C 1 -C 20 alkyl 5 -C 20 -heteroaryl radical.
  • the alkyl radical may be saturated, mono- or polyunsaturated, linear, branched or cyclic.
  • the aryl radicals and heteroaryl radicals are as defined above.
  • R 3 is preferably a C 1 -C 12 -alkyl radical. Suitable substituents are, for example, phenyl, C 1 -C 6 -alkyl, OH, halogen or a sulfoxy group.
  • nitriles of the formula (III) are methyl cyanoformate, ethyl ester, 2,2,2-trichloroethyl ester, tert. butyl ester, benzyl ester, allyl ester, -i-butyl ester, 2-ethylhexyl ester, p-menthyl ester, etc
  • Carbonic acid nitriles of the formula (III) are commercially available or can be prepared, for example, from the corresponding halides and HCN or an alkali metal cyanide, as described, for example, in EP 0 136 145, Tetrahedron Letters No. 2,829,165. 27, p. 2517 or J. Chem. Soc. Perkin Trans. 1, (15), 1729-35, 1993.
  • At least 1 mol, preferably from 1 to 5 mols, particularly preferably from 2 to 4 mols, of carbonitrile are added per mole of aldehyde or keto group used.
  • the reaction according to the invention takes place in the organic, aqueous or 2-phase system or in emulsion in the presence of a hydroxynitrile lyase as catalyst.
  • an aqueous solution or buffer solution containing the corresponding HNL is used in the aqueous system.
  • examples for this are acetate buffer, borate buffer, phthalate buffer, citrate buffer, phosphate buffer, etc, or mixtures of these buffer solutions.
  • the pH of this solution is at pH 2 to 8, preferably at pH 2.5 to 6.5, are.
  • organic diluent water-immiscible or slightly-miscible aliphatic or aromatic hydrocarbons optionally halogenated, alcohols, ethers or esters or mixtures thereof may be used. Preference is given to methyl tert. Butyl ether (MTBE), diisopropyl ether, dibutyl ether and ethyl acetate or mixtures thereof.
  • MTBE methyl tert. Butyl ether
  • diisopropyl ether dibutyl ether and ethyl acetate or mixtures thereof.
  • the reaction can also be carried out in a two-phase system or in emulsion.
  • Suitable HNLs are both native and recombinant (R) and (S) -HNLs, which are present either as such or immobilized.
  • native (S) -hydroxynitrile lyases for example from manioc and Hevea brasiliensis, as well as recombinant (S) -HNL are suitable.
  • Suitable recombinant (S) -HNL is obtained, for example, from genetically modified microorganisms such as Pichia pastoris, E. coli or Saccharomyces cerevisiae.
  • Preference is given to using recombinant (S) -Hnl from Pichia pastoris.
  • (R) -HNL for example, (R) -hydroxynitrile lyases from Prunus amygdalus, Prunus laurocerasus or Prunus serotina, or recombinant (R) -Hnl in question. Preference is given to using (R) -hydroxynitrilase from Prunus amygdalus or a recombinant (R) -HNL.
  • Suitable (R) and (S) -HNLs are described, for example, in WO 97/03204; EP 0 969 095; EP 0 951 561, EP 0 927 766, EP 0 632 130, EP 0547 655, EP 0 326 063, WO 01/44487, etc.
  • hydroxynitrile lyase is added per g of aldehyde or ketone.
  • reaction temperatures are about -5 to + 40 ° C, preferably about 0 to 30 ° C.
  • the reaction according to the invention is carried out in an aqueous system, wherein the corresponding HNL is initially introduced as an aqueous solution, depending on the selected HNL by means of a suitable acid, for example by means of citric acid or with a buffer, such as acetate buffer, borate buffer, phthalate buffer, citrate buffer, phosphate buffer etc, or mixtures of these buffer solutions, to the desired pH.
  • a suitable acid for example by means of citric acid or with a buffer, such as acetate buffer, borate buffer, phthalate buffer, citrate buffer, phosphate buffer etc, or mixtures of these buffer solutions, to the desired pH.
  • a buffer such as acetate buffer, borate buffer, phthalate buffer, citrate buffer, phosphate buffer etc, or mixtures of these buffer solutions
  • HCN evolves under HNL-catalyzed addition with the starting material first to a corresponding enantioenriched cyanohydrin.
  • Residual carbonic acid nitrile reacts with the enantiomerically enriched cyanohydrin to form the stable, O-protected, enantiomerically enriched cyanohydrin of formula (I), again releasing HCN, which in turn is used for cyanohydrin formation.
  • HCN enantiomerically enriched cyanohydrin
  • the process according to the invention makes it possible by the chemical O-derivatization to shift the equilibrium to the side of the desired end product, whereby a significantly higher conversion can be achieved in particular with cyanohydrins having an initially unfavorable equilibrium position, such as acetophenone derivatives, compared to the prior art. At the same time stabilization of the cyanohydrins formed is achieved by the derivatization.
  • a further advantage of the method according to the invention is the in situ generation of HCN and the continuous delivery of HCN, at the same time avoiding the direct onset of HCN. Unexpectedly, the derivatization reagent does not or only insignificantly lowers the activity of the HNL used.

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Abstract

Production of protected enantiomer-enriched cyanohydrins (I) comprises reacting an aldehyde or ketone (II) with a cyanoformate ester (III) in the presence of an (R)- or (S)-hydroxynitrile lyase in an organic, aqueous, 2-phase or emulsion medium at -5 to +40 degrees C. <??>Production of protected enantiomer-enriched cyanohydrins of formula NCC(R1)(R2)OC(O)OR3 (I) comprises reacting an aldehyde or ketone of formula R1C(O)R2 (II) with a cyanoformate ester of formula NCC(O)OR3 (III) in the presence of an (R)- or (S)-hydroxynitrile lyase in an organic, aqueous, 2-phase or emulsion medium at -5 to +40 degrees C. <??>R1, R2 = H or optionally substituted 1-20C alkyl, 5-20C aryl, 5-20C heteroaryl, 5-20C alkaryl, 5-20C alkylheteroaryl or 5-20C aralkyl; or <??>R1+R2 = optionally substituted 4-20C alkylene or heteroalkylene; and <??>R3 = optionally substituted 1-20C alkyl, 5-20C aryl or 5-20C heteroaryl; <??>provided that R1 and R2 are not both H.

Description

Cyanhydrine sind etwa zur Synthese von alpha-Hydroxysäuren, alpha-Hydroxyketonen, beta-Aminoalkoholen, die zur Gewinnung biologisch wirksamer Stoffe, z. B. pharmazeutischer Wirkstoffe, Vitamine oder auch pyrethroider Verbindungen Verwendung finden, von Bedeutung.Cyanohydrins are for example for the synthesis of alpha-hydroxy acids, alpha-hydroxy ketones, beta-amino alcohols, which are used to obtain biologically active substances, eg. As pharmaceutical agents, vitamins or pyrethroid compounds find use of importance.

Die Herstellung eines Cyanhydrins kann durch Anlagerung von Blausäure (HCN) an die Carbonylgruppe eines Aldehyds oder eines Ketons erfolgen, wobei Enantiomerengemische unsymmetrischer Cyanhydrine entstehen.
Viele Verfahren beruhen darauf, die Anlagerung von HCN an die Carbonylgruppe in Gegenwart eines chiralen Katalysators, beispielsweise einer Hydroxynitrillyase durchzuführen.
Da HCN jedoch eine äußerst giftige Substanz ist, wird ständig versucht den direkten Einsatz bzw. das direkte Handling zu vermeiden. Als Alternativen zu HCN wurden bisher beispielsweise Cyanhydrine der allgemeinen Formel RR'C(OH)(CN), wobei R und R' unabhängig voneinander Wasserstoff oder eine unsubstituierte Kohlenwasserstoffgruppe, oder gemeinsam eine Alkylengruppe mit 4 oder 5 C-Atomen, wobei R und R' nicht gleichzeitig Wasserstoff bedeuten, wie etwa Acetocyanhydrin, als Cyanidgruppendonor eingesetzt.
Einen weiteren Cyanidgruppendonor stellt beispielsweise Trimethylsilylcyanid dar, der gemäß J. Am. Chem. Soc. 2001, 123, 9908-9909 mit Zuckerderivaten bei -40°C in einem absoluten Alkohol umgesetzt wird.The preparation of a cyanohydrin can be carried out by addition of hydrogen cyanide (HCN) to the carbonyl group of an aldehyde or a ketone, resulting in enantiomeric mixtures of asymmetric cyanohydrins.
Many methods are based on carrying out the addition of HCN to the carbonyl group in the presence of a chiral catalyst, for example a hydroxynitrile lyase.
However, since HCN is a very toxic substance, it is constantly trying to avoid direct use or direct handling. Cyanohydrins of the general formula RR'C (OH) (CN), where R and R 'independently of one another are hydrogen or an unsubstituted hydrocarbon group, or together have an alkylene group with 4 or 5 C atoms, where R and R 'not simultaneously hydrogen, such as acetocyanohydrin, used as a cyanide donor.
Another cyanide group donor is, for example, trimethylsilyl cyanide, which according to J. Am. Chem. Soc. 2001, 123, 9908-9909 is reacted with sugar derivatives at -40 ° C in an absolute alcohol.

Ein weiteres Problem in der Herstellung von Cyanhydrinen ist, dass Cyanhydrine an sich unbeständig sind und dazu neigen, sich in Umkehr ihrer Bildungsreaktion zu zersetzen, sodass bereits versucht wurde diese durch die unterschiedlichsten Zusätze, insbesondere von Säuren, wie etwa Schwefelsäure, Phosphorsäure, HCI, Toluolsulfonsäure, Essigsäure, Propionsäure u.s.w. zu stabilisieren.Another problem in the production of cyanohydrins is that cyanohydrins are unstable per se and tend to decompose in reversal of their formation reaction, so they have already been tried by the various additives, in particular of acids such as sulfuric acid, phosphoric acid, HCl, toluenesulfonic acid, acetic acid, propionic acid, etc.

Bei manchen Cyanhydrinen, wie etwa bei Acetophenonderivaten, ist außerdem die Gleichgewichtslage der Reaktion ziemlich ungünstig, wodurch diese Cyanhydrine nur in schlechten Ausbeuten erhalten werden.In addition, for some cyanohydrins, such as acetophenone derivatives, the equilibrium position of the reaction is rather unfavorable, resulting in these cyanohydrins being obtained only in poor yields.

Aufgabe der vorliegenden Erfindung war es, ein Hydroxynitrillyase-katalysiertes Verfahren zur Herstellung von stabilen, enantiomerenangereicherten Cyanhydrinen zu finden, bei welchem der direkte Einsatz von Blausäure vermieden wird und das eine Gleichgewichtsverschiebung zur Erzielung hoher Konversionen ermöglicht.The object of the present invention was to find a hydroxynitrile lyyl catalyzed process for the preparation of stable, enantiomerically enriched cyanohydrins in which the direct use of hydrogen cyanide is avoided and which allows a shift in equilibrium to achieve high conversions.

Unerwarteterweise konnte diese Aufgabe durch ein Verfahren gelöst werden, bei welchem Kohlensäureesternitrile als Cyanidgruppendonoren eingesetzt werden, wodurch eine in-situ-Derivatisierung und somit Stabilisierung der enantiomerenangereicherten Cyanhydrine erfolgt.Unexpectedly, this object could be achieved by a method in which carbonic acid nitriles are used as cyanide group donors, whereby an in situ derivatization and thus stabilization of the enantiomerically enriched cyanohydrins occurs.

Gegenstand der vorliegenden Erfindung ist demnach ein Verfahren zur Herstellung von geschützten, enantiomeren-angereicherten Cyanhydrinen der Formel

Figure imgb0001
in der R1 und R2 unabhängig voneinander einen gegebenenfalls ein- oder mehrfach substituierten C1-C20-Alkyl-, C5-C20-Aryl-, C5-C20-Heteroaryl-, C5-C20-Alkaryl- C5-C20-Alkylheteroaryl- oder C5-C20-Aralkylrest oder einen gegebenenfalls ein- oder mehrfach substituierten C5-C20-Heterocyclus oder C5-C20-Alkylheterocyclus oder gemeinsam einen gegebenenfalls substituierten C4-C20-Alkylenrest, der ein oder mehrere Heteroatome in der Kette enthalten kann bedeuten können, oder einer der Reste Wasserstoff bedeutet, und R3 ein gegebenenfalls substituierter C1-C20-Alkyl-, C5-C20-Aryl- oder C5-C20-Heteroarylrest sein kann, das dadurch gekennzeichnet ist, dass ein Aldehyd oder Keton der Formel
Figure imgb0002
 in der R1 und R2 wie oben definiert sind, in Gegenwart einer (R)- oder (S)-Hydroxynitrillyase im organischen, wässrigen oder 2-Phasensystem oder in Emulsion bei einer Temperatur von -5 bis +40°C mit einem Kohlensäureesternitril der Formel
Figure imgb0003
 in der R3 wie oben definiert ist,
zu den entsprechenden O-geschützten, enantiomeren-angereicherten Cyanhydrinen der Formel (I) umgesetzt werden.The present invention accordingly provides a process for the preparation of protected, enantiomerically enriched cyanohydrins of the formula
Figure imgb0001
 in the R 1 and R 2, independently of one another, an optionally mono- or polysubstituted C 1 -C 20 -alkyl, C 5 -C 20 -aryl, C 5 -C 20 -heteroaryl, C 5 -C 20 -alkaryl-C C 5 -C 20 -alkylheteroaryl or C 5 -C 20 -aralkyl radical or an optionally mono- or polysubstituted C 5 -C 20 -heterocycle or C 5 -C 20 -alkyl heterocycle or together an optionally substituted C 4 -C 20 -alkylene radical which may mean one or more heteroatoms in the chain, or one of the radicals Is hydrogen, and R3 may be an optionally substituted C 1 -C 20 alkyl, C 5 -C 20 aryl or C 5 -C 20 heteroaryl radical, which is characterized in that an aldehyde or ketone of the formula
Figure imgb0002
 in which R 1 and R 2 are as defined above, in the presence of an (R) - or (S) -hydroxynitrile lyase in the organic, aqueous or 2-phase system or in emulsion at a temperature of -5 to + 40 ° C with a carbonic acid nitrile of the formula
Figure imgb0003
 where R3 is as defined above
to the corresponding O-protected, enantiomerically-enriched cyanohydrins of the formula (I) are reacted.

Bei dem erfindungsgemäßen Verfahren werden Aldehyde oder Ketone der Formel (II) als Edukte verwendet.
In der Formel (II) können R1 und R2 unabhängig voneinander einen gegebenenfalls ein- oder mehrfach substituierten C1-C20-Alkyl-, C5-C20-Aryl-, C5-C20-Heteroaryl-, C5-C20-Alkaryl-, C5-C20- Alkylheteroaryl- oder C5-C20-Aralkylrest oder einen gegebenenfalls ein- oder mehrfach substituierten C5-C20-Heterocyclus oder C5-C20-Alkylheterocyclus bedeuten.In the process according to the invention, aldehydes or ketones of the formula (II) are used as starting materials.
In the formula (II) R1 and R2 are each independently an unsubstituted, monosubstituted or polysubstituted C 1 -C 20 alkyl, C 5 -C 20 aryl, C 5 -C 20 -heteroaryl, C 5- C 20 -Alkaryl-, C 5 -C 20 - Alkylheteroaryl- or C 5 -C 20 -Aralkylrest or an optionally mono- or polysubstituted C 5 -C 20 heterocycle or C 5 -C 20 alkylheterocycle.

Unter C1-C20-Alkyl sind dabei gesättigte oder ein- oder mehrfach ungesättigte, lineare, verzweigte oder cyclische, primäre, sekundäre oder tertiäre Hydrocarbonreste zu verstehen. Dies sind beispielsweise C1-C20-Alkylreste, wie etwa Methyl, Ethyl, Propyl, iso-Propyl, Butyl, iso-Butyl, t-Butyl, Butenyl, Butinyl, Pentyl, Cyclopentyl, iso-Pentyl, neo-Pentyl, Pentenyl, Pentinyl, Hexyl, iso-Hexyl, Cyclohexyl, Cyclohexylmethyl, 3-Methylpentyl, 2,2-Dimethylbutyl, 2,3-Dimethylbutyl, Octyl, Cyclooctyl, Decyl, Cyclodecyl, Dodecyl, Cyclododecyl u.s.w. Bevorzugt sind dabei C1-C12-Alkylreste und besonders bevorzugt C1-C8-Alkylreste. Die Alkylgruppe kann gegebenenfalls ein- oder mehrfach durch unter den Reaktionsbedingungen inerte Gruppen substituiert sein. Geeignete Substituenten sind beispielsweise gegebenenfalls substituierte Aryl- oder Heteroarylgruppen, wie Phenyl-, Phenoxy- oder Indolylgruppen, Halogen-, Hydroxy-, Hydroxy-C1-C5-Alkyl, C1-C6-Alkoxy-, Aryloxy-, bevorzugt C6-C20-Aryloxy-, C1-C6-Alkylthio-,- Amino-, Alkylamino-, bevorzugt C1-C6-Alkylamino-, Arylamino-, bevorzugt C6-C20-Arylamino-, Ether-, Thioether, Carbonsäureester-, Carbonsäureamid-, Sulfoxid-, Sulfon-, Sulfonsäure, Sulfonsäureester-, Sulfinsäure-,Mercaptan-, Nitro- oder Azidogruppen.By C 1 -C 20 -alkyl are meant saturated or mono- or polyunsaturated, linear, branched or cyclic, primary, secondary or tertiary hydrocarbon radicals. These are, for example, C 1 -C 20 -alkyl radicals, such as, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, butenyl, butynyl, pentyl, cyclopentyl, isopentyl, neopentyl, pentenyl , Pentynyl, hexyl, isohexyl, cyclohexyl, cyclohexylmethyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, octyl, cyclooctyl, decyl, cyclodecyl, dodecyl, cyclododecyl, etc. Preferred are C 1 -C 12 -alkyl radicals and particularly preferably C 1 -C 8 -alkyl radicals. The alkyl group may optionally be monosubstituted or polysubstituted by groups inert under the reaction conditions. Suitable substituents are, for example, optionally substituted aryl or heteroaryl groups, such as phenyl, phenoxy or indolyl groups, halogen, hydroxy, hydroxy-C 1 -C 5 -alkyl, C 1 -C 6 -alkoxy, aryloxy, preferably C C 6 -C 20 -aryloxy, C 1 -C 6 -alkylthio, -amino, alkylamino, preferably C 1 -C 6 -alkylamino, arylamino, preferably C 6 -C 20 -arylamino, ether, Thioethers, carboxylic acid ester, carboxylic acid amide, sulfoxide, sulfonic, sulfonic, sulfonic acid, sulfinic, mercaptan, nitro or azido groups.

Unter Aryl sind bevorzugt C6-C20-Arylgruppen zu verstehen, wie etwa Phenyl, Biphenyl, Naphthyl, Indenyl, Fluorenyl u.s.w.Aryl is preferably C 6 -C 20 -aryl groups, such as phenyl, biphenyl, naphthyl, indenyl, fluorenyl, etc

Die Arylgruppe kann dabei gegebenenfalls ein- oder mehrfach substituiert sein. Geeignete Substituenten sind dabei wiederum gegebenenfalls substituierte Aryl- oder Heteroarylgruppen, wie Phenyl-, Phenoxy- oder Indolylgruppen, Halogen-, Hydroxy-, Hydroxy-C1-C5-Alkyl, C1-C6-Alkoxy-, Aryloxy-, bevorzugt C6-C20-Aryloxy-, C1-C6-Alkylthio-, Amino-, Alkylamino-, bevorzugt C1-C6-Alkylamino-, Arylamino-, bevorzugt C6-C20-Arylamino-, Ether-, Thioether, Carbonsäureester-, Carbonsäureamid-, Sulfoxid-, Sulfon-, Sulfonsäure, Sulfonsäureester-, Sulfinsäure-,Mercaptan-, Nitro- oder Azidogruppen.The aryl group may optionally be monosubstituted or polysubstituted. Suitable substituents are in turn optionally substituted aryl or heteroaryl groups such as phenyl, phenoxy or indolyl, halogen, hydroxy, hydroxy-C 1 -C 5 alkyl, C 1 -C 6 alkoxy, aryloxy, preferred C 6 -C 20 -aryloxy, C 1 -C 6 -alkylthio, amino, alkylamino, preferably C 1 -C 6 -alkylamino, arylamino, preferably C 6 -C 20 -arylamino, ether, Thioethers, carboxylic acid ester, carboxylic acid amide, sulfoxide, sulfonic, sulfonic, sulfonic acid, sulfinic, mercaptan, nitro or azido groups.

Unter Alkaryl oder Alkylaryl sind Alkylgruppen zu verstehen, die einen Arylsubstituenten aufweisen.
Aralkyl oder Arylalkyl bezieht sich auf eine Arylgruppe mit einem Alkylsubstituenten.By alkaryl or alkylaryl are meant alkyl groups having an aryl substituent.
Aralkyl or arylalkyl refers to an aryl group having an alkyl substituent.

Unter Heteroaryl oder Heterocyclus sind cyclische Reste zu verstehen die zumindestens ein S-, O- oder N-Atom im Ring enthalten. Dies sind beispielsweise Furyl, Pyridyl, Pyrimidyl, Thienyl, Isothiazolyl, Imidazolyl, Tetrazolyl, Pyrazinyl, Benzofuranyl, Benzothiophenyl, Chinolyl, Isochinolyl, Benzothienyl, Isobenzofuryl, Pyrazolyl, Indolyl, Isoindolyl, Benzoimidazolyl, Purinyl, Carbazolyl, Oxazolyl, Thiazolyl, Isothiazolyl, 1,2,4-Thiadiazolyl, Isoxazolyl, Pyrrolyl, Chinazolinyl, Pyridazinyl, Phthalazinyl, Morpholinyl, u.s.w.
Funktionelle O- oder N-Gruppen können dabei nötigenfalls geschützt werden.
Die Heteroarylgruppe bzw. der Heterocyclus kann dabei gegebenenfalls ein- oder mehrfach durch die bereits oben angeführten Substituenten substituiert sein.By heteroaryl or heterocycle are meant cyclic radicals containing at least one S, O or N atom in the ring. These are, for example, furyl, Pyridyl, pyrimidyl, thienyl, isothiazolyl, imidazolyl, tetrazolyl, pyrazinyl, benzofuranyl, benzothiophenyl, quinolyl, isoquinolyl, benzothienyl, isobenzofuryl, pyrazolyl, indolyl, isoindolyl, benzoimidazolyl, purinyl, carbazolyl, oxazolyl, thiazolyl, isothiazolyl, 1,2,4- Thiadiazolyl, isoxazolyl, pyrrolyl, quinazolinyl, pyridazinyl, phthalazinyl, morpholinyl, etc
Functional O or N groups can be protected if necessary.
The heteroaryl group or the heterocycle may optionally be monosubstituted or polysubstituted by the abovementioned substituents.

Unter Alkylheteroaryl bzw. Alkylheterocyclus sind dabei Alkylgruppen zu verstehen, die durch eine Heteroarylgruppe bzw. durch einen Heterocyclus substituiert sind.By alkyl heteroaryl or alkyl heterocycle are meant alkyl groups which are substituted by a heteroaryl group or by a heterocycle.

Bevorzugt bedeuten R1 und R2 einen gesättigten, linearen oder verzweigten C1-C8-Alkylrest, einen Benzyl- oder einen Phenylrest, wobei die Reste gegebenenfalls ein- oder mehrfach durch F, Cl, OH, Carbonsäurederivate, wie Carbonsäureester oder Carbonsäureamide, Amino, C1-C6-Alkylamino, C6-C20-Arylamino, C1-C6-Alkoxy, C6-C20-Aryloxy, oder Nitro substituiert sein können.R 1 and R 2 preferably denote a saturated, linear or branched C 1 -C 8 -alkyl radical, a benzyl radical or a phenyl radical, where the radicals are optionally mono- or polysubstituted by F, Cl, OH, carboxylic acid derivatives, such as carboxylic acid esters or carboxamides, amino, C 1 -C 6 alkylamino, C 6 -C 20 arylamino, C 1 -C 6 alkoxy, C 6 -C 20 aryloxy, or nitro may be substituted.

R1 und R2 können aber auch gemeinsam einen gegebenenfalls substituierten C4-C20-Alkylenrest, der ein oder mehrere Heteroatome aus der Gruppe O, N oder S oder eine NR4R5-Gruppe, wobei R4 und R5 unabhängig voneinander H oder C1-C6-Alkylsein können, in der Kette enthalten kann bedeuten. In diesem Fall stellen die Edukte cyclische Ketone dar.
Bevorzugt sind C4-C7-Alkylenreste, die in Abhängigkeit von der Ringgröße des cyclischen Ketons höchstens 2 Heteroatome in der Alkylkette aufweisen. Der Alkylenrest kann weiters noch, wiederum in Abhängigkeit von der Ringgröße des cyclischen Ketons noch ein oder 2 Doppelbindungen aufweisen, wobei bei einem 5er-Ring diese nicht in Konjugation zur Carbonylgruppe stehen darf.
Der Alkylenrest kann zudem ein oder mehrfach durch die oben angeführten Reste substituiert sein.However, R 1 and R 2 together may also be an optionally substituted C 4 -C 20 -alkylene radical which contains one or more heteroatoms from the group consisting of O, N or S or an NR 4 R 5 -group, where R 4 and R 5 independently of one another are H or C 1 -C 6 - Alkyl may be included in the chain. In this case, the starting materials are cyclic ketones.
Preference is given to C 4 -C 7 -alkylene radicals which, depending on the ring size of the cyclic ketone, have at most 2 heteroatoms in the alkyl chain. The alkylene radical can still, in turn, depending on the ring size of the cyclic ketone still have one or two double bonds, which in a 5-membered ring may not be in conjugation to the carbonyl group.
The alkylene radical may also be substituted one or more times by the radicals mentioned above.

Bei den eingesetzten Edukten kann jedoch auch einer der Reste R1 und R2 Wasserstoff bedeuten. In diesem Fall handelt es sich bei den Edukten um Aldehyde.In the case of the educts used, however, one of the radicals R 1 and R 2 may also be hydrogen. In this case the educts are aldehydes.

Erfindungsgemäß wird das gewünschte Edukt mit einem Kohlensäureesternitril der Formel (III) umgesetzt.
In der Formel (III) bedeutet R3 einen gegebenenfalls substituierten C1-C20-Alkyl-, C5-C20-Aryl- oder C5-C20-Heteroarylrest. Der Alkylrest kann dabei gesättigt, ein- oder mehrfach ungesättigt, linear, verzweigt oder cyclisch sein. Die Arylreste und Heteroarylreste sind dabei wie oben definiert. Bevorzugt bedeutet R3 einen C1-C12-Alkylrest.
Geeignete Substituenten sind beispielsweise Phenyl, C1-C6-Alkyl, OH, Halogen oder eine Sulfoxygruppe.
Beispiele für geeignete Nitrile der Formel (III) sind Cyanameisensäuremethylester, -ethylester, -2,2,2-trichlorethylester, -tert. butylester, -benzylester, -allylester, -i-butylester, -2-ethylhexylester, -p-menthylester, u.s.w.According to the invention, the desired educt is reacted with a carbonic acid nitrile of the formula (III).
In the formula (III) R3 is an optionally substituted C, C 5- C 20 aryl or C 1 -C 20 alkyl 5 -C 20 -heteroaryl radical. The alkyl radical may be saturated, mono- or polyunsaturated, linear, branched or cyclic. The aryl radicals and heteroaryl radicals are as defined above. R 3 is preferably a C 1 -C 12 -alkyl radical.
Suitable substituents are, for example, phenyl, C 1 -C 6 -alkyl, OH, halogen or a sulfoxy group.
Examples of suitable nitriles of the formula (III) are methyl cyanoformate, ethyl ester, 2,2,2-trichloroethyl ester, tert. butyl ester, benzyl ester, allyl ester, -i-butyl ester, 2-ethylhexyl ester, p-menthyl ester, etc

Kohlensäureesternitrile der Formel (III) sind käuflich erwerbbar oder können beispielsweise aus den entsprechenden Halogeniden und HCN oder einem Alkalicyanid, wie etwa in EP 0 136 145, Tetrahedron Letters No. 27, S.2517 oder J.Chem. Soc. Perkin Trans. 1, (15), 1729-35, beschrieben, 1993 hergestellt werden.Carbonic acid nitriles of the formula (III) are commercially available or can be prepared, for example, from the corresponding halides and HCN or an alkali metal cyanide, as described, for example, in EP 0 136 145, Tetrahedron Letters No. 2,829,165. 27, p. 2517 or J. Chem. Soc. Perkin Trans. 1, (15), 1729-35, 1993.

Pro Mol eingesetzte Aldehyd- oder Ketogruppe werden mindestens 1 Mol, bevorzugt 1 bis 5 Mole, besonders bevorzugt 2 bis 4 Mole, Kohlensäurenitril zugegeben.At least 1 mol, preferably from 1 to 5 mols, particularly preferably from 2 to 4 mols, of carbonitrile are added per mole of aldehyde or keto group used.

Die erfindungsgemäße Reaktion findet im organischen, wässrigen oder 2-Phasensystem oder in Emulsion in Anwesenheit einer Hydroxynitrillyase als Katalysator statt.
Dabei wird bei der enantioselektiven Umsetzung im wässrigen System eine wässrige, die entsprechende HNL enthaltende Lösung oder Pufferlösung verwendet. Beispiele dafür sind Acetatpuffer, Boratpuffer, Phthalatpuffer, Citratpuffer, Phosphatpuffer u.s.w. oder Gemischen dieser Pufferlösungen.
Der pH-Wert dieser Lösung liegt dabei bei pH 2 bis 8, bevorzugt bei pH 2,5 bis 6,5, liegen.The reaction according to the invention takes place in the organic, aqueous or 2-phase system or in emulsion in the presence of a hydroxynitrile lyase as catalyst.
In the enantioselective reaction, an aqueous solution or buffer solution containing the corresponding HNL is used in the aqueous system. Examples for this are acetate buffer, borate buffer, phthalate buffer, citrate buffer, phosphate buffer, etc, or mixtures of these buffer solutions.
The pH of this solution is at pH 2 to 8, preferably at pH 2.5 to 6.5, are.

Als organisches Verdünnungsmittel können mit Wasser nicht oder geringfügig mischbare aliphatische oder aromatische Kohlenwasserstoffe, die gegebenenfalls halogeniert sind, Alkohole, Ether oder Ester oder Gemische davon verwendet werden. Bevorzugt werden Methyl -tert. Butylether (MTBE), Diisopropylether, Dibutylether und Ethylacetat oder deren Gemische eingesetzt.
Die Umsetzung kann jedoch auch in einem Zweiphasensystem oder in Emulsion mit erfolgen.As the organic diluent, water-immiscible or slightly-miscible aliphatic or aromatic hydrocarbons optionally halogenated, alcohols, ethers or esters or mixtures thereof may be used. Preference is given to methyl tert. Butyl ether (MTBE), diisopropyl ether, dibutyl ether and ethyl acetate or mixtures thereof.
However, the reaction can also be carried out in a two-phase system or in emulsion.

Als HNLs eignen sich sowohl native als auch rekombinante (R)- und (S)-HNLs, die entweder als solche oder immobilisiert vorliegen.Suitable HNLs are both native and recombinant (R) and (S) -HNLs, which are present either as such or immobilized.

Als (S)-Hydroxynitrillyase (HNL) kommen native (S)-Hydroxynitrillyasen z.B. aus Maniok und Hevea brasiliensis, sowie rekombinante (S)-HNL in Frage. Bevorzugt wird als native HNL HNL aus Hevea brasiliensis verwendet. Geeignete rekombinante (S)-HNL wird beispielsweise aus gentechnisch modifizierten Mikroorganismen, wie etwa Pichia pastoris, E. coli oder Saccharomyces cerevisiae erhalten.
Bevorzugt wird rekombinante (S)-Hnl aus Pichia pastoris eingesetzt.As (S) -hydroxynitrile lyase (HNL), native (S) -hydroxynitrile lyases, for example from manioc and Hevea brasiliensis, as well as recombinant (S) -HNL are suitable. Preference is given to using native HNL HNL from Hevea brasiliensis. Suitable recombinant (S) -HNL is obtained, for example, from genetically modified microorganisms such as Pichia pastoris, E. coli or Saccharomyces cerevisiae.
Preference is given to using recombinant (S) -Hnl from Pichia pastoris.

Als (R)-HNL kommen beispielsweise (R)-Hydroxynitrillyasen aus Prunus amygdalus, Prunus laurocerasus oder Prunus serotina, oder rekombinante (R)-Hnl in Frage. Bevorzugt wird (R)-Hydroxynitrilase aus Prunus amygdalus oder eine rekombinante (R)-HNL verwendet.As (R) -HNL, for example, (R) -hydroxynitrile lyases from Prunus amygdalus, Prunus laurocerasus or Prunus serotina, or recombinant (R) -Hnl in question. Preference is given to using (R) -hydroxynitrilase from Prunus amygdalus or a recombinant (R) -HNL.

Geeignete (R)- und (S)-HNLs sind beispielsweise aus WO 97/03204; EP 0 969 095; EP 0 951 561, EP 0 927 766, EP 0 632 130, EP 0547 655, EP 0 326 063, WO 01/44487 usw. bekannt.Suitable (R) and (S) -HNLs are described, for example, in WO 97/03204; EP 0 969 095; EP 0 951 561, EP 0 927 766, EP 0 632 130, EP 0547 655, EP 0 326 063, WO 01/44487, etc.

Pro g Aldehyd bzw. Keton werden etwa 10 bis 20 000 IU Aktivität, bevorzugt etwa 100 bis 10 000 IU Aktivität, Hydroxynitrillyase zugesetzt.About 10 to 20,000 IU of activity, preferably about 100 to 10,000 IU of activity, hydroxynitrile lyase is added per g of aldehyde or ketone.

Die Reaktionstemperaturen liegt bei etwa -5 bis +40°C, bevorzugt bei etwa 0 bis 30°C.The reaction temperatures are about -5 to + 40 ° C, preferably about 0 to 30 ° C.

Bevorzugt wird die erfindungsgemäße Reaktion im wässrigen System durchgeführt, wobei zuerst die entsprechende HNL als wässrige Lösung vorgelegt, in Abhängigkeit von der gewählten HNL mittels einer geeigneten Säure, beispielsweise mittels Citronensäure oder mit einem Puffer, wie etwa Acetatpuffer, Boratpuffer, Phthalatpuffer, Citratpuffer, Phosphatpuffer u.s.w. oder Gemischen dieser Pufferlösungen, auf den gewünschten pH gestellt wird. Anschließend wird das entsprechende Edukt der Formel (II) zugesetzt und die Reaktion durch Zugabe des Kohlensäureesternitrils der Formel (III) gestartet. Dabei entwickelt sich HCN das unter HNL-katalysierter Addition mit dem eingesetzten Edukt zuerst zu einem korrespondierenden enantiomerenangereicherten Cyanhydrin reagiert. Restliches Kohlensäurenitril reagiert mit dem enantiomerenangereicherten Cyanhydrin zu dem stabilen, O-geschützten, enantiomerenangereicherten Cyanhydrin der Formel (I), wobei wieder HCN frei wird, die wiederum zur Cyanhydrinbildung verwendet wird.
Es kann jedoch auch zuerst das Edukt vorgelegt werden und anschließend die entsprechende HNL als wässrige Lösung zugegeben werden.Preferably, the reaction according to the invention is carried out in an aqueous system, wherein the corresponding HNL is initially introduced as an aqueous solution, depending on the selected HNL by means of a suitable acid, for example by means of citric acid or with a buffer, such as acetate buffer, borate buffer, phthalate buffer, citrate buffer, phosphate buffer etc, or mixtures of these buffer solutions, to the desired pH. Subsequently, the corresponding starting material of the formula (II) is added and the reaction is started by adding the carbonic acid nitrile of the formula (III). HCN evolves under HNL-catalyzed addition with the starting material first to a corresponding enantioenriched cyanohydrin. Residual carbonic acid nitrile reacts with the enantiomerically enriched cyanohydrin to form the stable, O-protected, enantiomerically enriched cyanohydrin of formula (I), again releasing HCN, which in turn is used for cyanohydrin formation.
However, it is also possible first to introduce the educt and then to add the corresponding HNL as an aqueous solution.

Der Reaktionsverlauf ist aus folgendem Schema ersichtlich:

Figure imgb0004
The course of the reaction is shown in the following scheme:
Figure imgb0004

Das erfindungsgemäße Verfahren ermöglicht dabei durch die chemische O-Derivatisierung eine Gleichgewichtsverschiebung auf die Seite des gewünschten Endproduktes, wodurch insbesondere bei Cyanhydrinen mit ursprünglich ungünstiger Gleichgewichtslage, wie etwa Acetophenonderivate, im Vergleich zum Stand der Technik eine wesentlich höhere Konversion erzielt werden kann. Gleichzeitig wird durch die Derivatisierung eine Stabilisierung der gebildeten Cyanhydrine erreicht. Ein weiterer Vorteil des erfindungsgemäßen Verfahrens ist die in situ Generierung von HCN und die ständige Nachlieferung von HCN, wobei gleichzeitig ein direktes Einsetzen von HCN vermieden wird. Das Derivatisierungsreagens setzt dabei unerwarteterweise die Aktivität der eingesetzten HNL nicht oder nur unbedeutend herab.The process according to the invention makes it possible by the chemical O-derivatization to shift the equilibrium to the side of the desired end product, whereby a significantly higher conversion can be achieved in particular with cyanohydrins having an initially unfavorable equilibrium position, such as acetophenone derivatives, compared to the prior art. At the same time stabilization of the cyanohydrins formed is achieved by the derivatization. A further advantage of the method according to the invention is the in situ generation of HCN and the continuous delivery of HCN, at the same time avoiding the direct onset of HCN. Unexpectedly, the derivatization reagent does not or only insignificantly lowers the activity of the HNL used.

Beispiel 1:Example 1: HNL-katalysierte Reaktion von Cyanameisensäureethylester mit Benzaldehyd:HNL-catalyzed reaction of ethyl cyanoformate with benzaldehyde:

Figure imgb0005
Figure imgb0005

Es wurden 2,5 ml rekombinante R-HNL Lösung (300 iU/ml) mit einer Citronensäurelösung auf pH 3,3 eingestellt und mit 2,5 ml 50 mmol Kaliumphosphat / Citratpuffer pH 3,3 verdünnt. Anschließend wurden 106 mg (1 mmol) Benzaldehyd zugesetzt und die Reaktion durch Zugabe von 297 µl (3 mmol) Cyanameisensäureethylester gestartet. Das Reaktionsgemisch wurde bei 25 °C gerührt und die Bildung von Benzaldehydcyanhydrin und O-Ethoxycarbonyl-cyanhydrin mittels Gaschromatographie an einer chiralen Phase (Cyclodextrinsäule) verfolgt und die entsprechenden Enantiomerenreinheiten errechnet.2.5 ml of recombinant R-HNL solution (300 iU / ml) was adjusted to pH 3.3 with a citric acid solution and diluted with 2.5 ml of 50 mM potassium phosphate / citrate buffer pH 3.3. Subsequently, 106 mg (1 mmol) of benzaldehyde were added and the reaction started by adding 297 μl (3 mmol) of ethyl cyanoformate. The reaction mixture was stirred at 25 ° C and the formation of Benzaldehydcyanhydrin and O-ethoxycarbonyl-cyanohydrin by gas chromatography on a chiral phase (cyclodextrin) followed and calculated the corresponding enantiomeric purities.

Reaktionsverlauf:Course of the reaction:

Reaktionszeit (Stunden) Benzaldehyd Benzaldehydcyanhydrin O-Ethoxycarbonyl-cyanhydrin (Area%) (Area%) (%ee) (Area%) (%ee) 1 61 38 99,9 1 99,9 3 15 77 99,5 8 99,9 23 2 65 93,7 33 99,9 44,5 * < 1 34 89,4 66 94,7 *Nach 26 Stunden wurden nochmals 297 µl (3 mmol) Cyanameisensäureethylester zugesetzt. Identifizierung des O-Ethoxycarbonyl-cyanhydrines:
1H-NMR in CDCl3, 300 MHz: δ 1,30-1,32 (t, 3H), 4,21-4,36 (m, 2H), 6,27 (s, 1H) 7,39-7,57 (m, 5H) Reaction time (hours) benzaldehyde benzaldehyde O-ethoxycarbonyl cyanohydrin (Area%) (Area%) (% Ee) (Area%) (% Ee) 1 61 38 99.9 1 99.9 3 15 77 99.5 8th 99.9 23 2 65 93.7 33 99.9 44.5 * <1 34 89.4 66 94.7 After 26 hours, another 297 μl (3 mmol) of ethyl cyanoformate were added. Identification of O-ethoxycarbonyl cyanohydrin:
1 H-NMR in CDCl 3 , 300 MHz: δ 1.30-1.32 (t, 3H), 4.21-4.36 (m, 2H), 6.27 (s, 1H) 7.39- 7.57 (m, 5H)

Claims (10)

  1. Process for preparing protected, enantiomer-enriched cyanohydrins of the formula
    Figure imgb0009
     where R1 and R2 independently of one another can be an unsubstituted, monosubstituted or polysubstituted C1-C20-alkyl, C5-C20-aryl, C5-C20-heteroaryl, C5-C20-alkaryl, C5-C20-alkylheteroaryl or C5-C20-aralkyl radical or an unsubstituted, monosubstituted or polysubstituted C5-C20-heterocycle, or C5-C20-alkylheterocycle or together can be an unsubstituted or substituted C4-C20-alkylene radical, which can contain one or more heteroatoms in the chain, or one of the radicals is hydrogen, and R3 can be an unsubstituted or substituted C1-C20-alkyl, C5-C20-aryl or C5-C20-heteroaryl radical, which process is characterized in that an aldehyde or ketone of the formula
    Figure imgb0010
     where R1 and R2 are defined as above, is reacted in the presence of an (R)- or (S)-hydroxynitrile lyase in an organic, aqueous or 2-phase system or in emulsion at a temperature of -5 to +40°C with a carbonic ester nitrile of the formula
    Figure imgb0011
     where R3 is defined as above,
    to give the corresponding O-protected, enantiomer-enriched cyanohydrins of the formula (I).
  2. Process according to Claim 1, characterized in that the starting materials used are compounds of the formula (II) where R1 and R2 independently of one another can be a C1-C20-alkyl, C5-C20-aryl, C5-C20-heteroaryl, C5-C20-alkaryl, C5-C20-alkylheteroaryl or C5-C20-aralkyl radical, or an unsubstituted, monosubstituted or polysubstituted C5-C20-heterocycle or C5-C20-alkylheterocycle or together can be an unsubstituted or substituted C4-C20-alkylene radical, which can contain one or more heteroatoms in the chain, where the radicals can be monosubstituted or polysubstituted by unsubstituted or substituted aryl or heteroaryl groups, halogen, hydroxyl, hydroxy-C1-C5-alkyl, C1-C6-alkoxy, C6-C20-aryloxy, C1-C6-alkylthio, amino, C1-C6-alkylamino, C6-C20-arylamino, ether, thioether, carboxylic ester, carboxamide, sulphoxide, sulphone, sulphonic acid, sulphonic ester, sulphinic acid, mercaptan, nitro or azido groups, or one of the radicals is hydrogen.
  3. Process according to Claim 2, characterized in that the starting materials used are compounds of the formula (II) where R1 and R2 independently of one another are a saturated, unbranched or branched C1-C8-alkyl radical, a benzyl radical or a phenyl radical, where the radicals can be unsubstituted, monosubstituted or polysubstituted by F, Cl, OH, carboxylic esters, carboxamides, amino, C1-C6-alkylamino, C6-C20-arylamino, C1-C6-alkoxy, C6-C20-aryloxy, or nitro, or one of the radicals is hydrogen.
  4. Process according to Claim 1, characterized in that the carbononitrile used is a compound of the formula (III) where R3 is a C1-C20-alkyl radical which can be substituted by one or more substituents selected from the group consisting of phenyl, C1-C6-alkyl, OH, halogen or sulphoxy.
  5. Process according to Claim 1, characterized in that, in the case of the enantioselective reaction in an aqueous system, an aqueous solution containing the corresponding hydroxynitrile lyase or an acetate buffer, borate buffer, phthalate buffer, citrate buffer, phosphate buffer solution or a mixture of these buffer solutions is used.
  6. Process according to Claim 5, characterized in that a pH of 2 to 8 is established in the aqueous solution.
  7. Process according to Claim 1, characterized in that, as organic diluent, water-immiscible or only slightly water-miscible aliphatic or aromatic hydrocarbons which may be halogenated, alcohols, ethers or esters or mixtures are used.
  8. Process according to Claim 1, characterized in that the reaction, however, alternatively proceeds in a two-phase system or in emulsion.
  9. Process according to Claim 1, characterized in that the hydroxynitrile lyases used are native or recombinant (R)- and (S)-hydroxynitrile lyases which are present either as such or immobilized.
  10. Process according to Claim 9, characterized in that the hydroxynitrile lyases used are native (S)-hydroxynitrile lyases from manioc and Hevea brasiliensis, recombinant (S)-hydroxynitrile lyase from genetically modified microorganisms from the group Pichia pastoris, E. coli or Saccharomyces cerevisiae, native (R)-hydroxynitrile lyases from Prunus amygdalus, Prunus laurocerasus or Prunus serotina, or recombinant (R)-hydroxynitrile lyases.
EP02026849A 2001-12-28 2002-11-29 Method for the preparation of protected, enantiomerically enriched cyanohydrines by in-situ derivatisation Expired - Lifetime EP1323829B1 (en)

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AT02026849T ATE328105T1 (en) 2001-12-28 2002-11-29 METHOD FOR PRODUCING PROTECTED, ENANTIOMER-ENriched CYANHYDRINS BY IN-SITU DERIVATIZATION

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AT20442001 2001-12-28
AT0204401A AT410792B (en) 2001-12-28 2001-12-28 PROCESS FOR THE PREPARATION OF PROTECTED, ENANTIOMERIC ENRICHED CYANHYDRINES BY IN SITU DERIVATIZATION

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EP1323829A2 EP1323829A2 (en) 2003-07-02
EP1323829A3 EP1323829A3 (en) 2003-12-10
EP1323829B1 true EP1323829B1 (en) 2006-05-31

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US (1) US6909011B2 (en)
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CA (1) CA2415190A1 (en)
DE (1) DE50206986D1 (en)
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DE10219934A1 (en) * 2002-05-03 2003-11-20 Basf Ag New proteins with (R) -hydroxynitrile lyase activity
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AT410792B (en) 2003-07-25
US20030129714A1 (en) 2003-07-10
CA2415190A1 (en) 2003-06-28
DE50206986D1 (en) 2006-07-06
EP1323829A2 (en) 2003-07-02
ATA20442001A (en) 2002-12-15
EP1323829A3 (en) 2003-12-10
ATE328105T1 (en) 2006-06-15
JP2003210187A (en) 2003-07-29
ES2262750T3 (en) 2006-12-01
US6909011B2 (en) 2005-06-21

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